This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. PI will be developing multiphoton (MPM) optical imaging methods to systematically study human glioblastoma multiform spheroids embedded in various 3D ECMs. These experimental cancer model systems are used to mimic the in vivo gliomas. An MPM ratiometric redox fluorometry method based on intrinsic cellular fluorescence from reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD) to study mitochondrial energy metabolism will be developed by the PI. Coupling these finding with the evaluations of mitochondrial membrane using a dye and visualizing F-actin distribution in conjunction with mitochondrial signals will furnish a better understanding on how ECM alters the growth, differentiation and metabolic states of the malignant glioma cells. The imaging methods developed will help cancer investigators to study key cancer properties such as cell survival and metastasis and can allow accurate measurements of tumor response to treatments. The studies proposed will benefit current therapies and prognosis for glioblastoma multiform.